HOXA10 regulates endometrial GABAA {pi} receptor expression and membrane translocation

Abstract

Expression of the GABA(A) pi receptor has been described previously in the human endometrium in both luminal epithelium and stroma. Its expression is increased during decidualization in rodents and in the implantation window of human endometrium. Here we localized GABA pi subunit receptor protein in human endometrium and identified regulators of gene expression and activation. GABA(A) pi was localized to the cell surface, and expression increased during the window of embryo implantation in human endometrium. The well-differentiated human endometrial adenocarcinoma cell line Ishikawa was treated with progesterone and transfected with pcDNA-HOXA10, HOXA10 siRNA, or respective controls. GABA(A) pi receptor mRNA expression was evaluated by real-time RT-PCR. Protein expression and localization were evaluated using immunofluorescence. GABA(A) pi receptor mRNA expression was increased significantly after either progesterone treatment or HOXA10 transfection. Coadministration of progesterone along with HOXA10 transfection had no additional effect on the expression of GABA(A) pi receptor mRNA over either agent alone. Blocking HOXA10 expression with siRNA prevented progesterone-induced GABA(A) pi receptor mRNA expression. Additionally, either HOXA10 or progesterone independently caused increased translocation of the GABA receptor from the cytoplasm to the cell membrane. Translocation in response to progesterone was blocked with HOXA10 siRNA. Progesterone-induced GABA(A) pi subunit receptor expression is likely mediated indirectly through progesterone's regulation of HOXA10 expression. Modification of subtype composition and translocation of the GABA receptor ion channel likely modulate endometrial receptivity. Whereas HOXA10 typically enhances the expression of progesterone-responsive genes, here HOXA10 expression leads to production of a less progestin-responsive GABA receptor subtype, likely buffering the effects of luteal phase progesterone on GABA receptor activity.

Fig. 1.

Immunohistochemical analysis of γ-aminobutyric acid (GABA)_A π receptor staining in endometrium on days 17 and 24, corresponding to the time of transition into the window of implantation. Day 17 (left) shows minimal stromal staining for GABA_A π receptor, and day 24 (right) shows increased positive staining for the receptor.

Fig. 2.

Regulation of GABA_A π receptor mRNA. There was a significant increase in mRNA levels of the receptor subunit in cells treated with progesterone (P), transfected with pcDNA-HOXA10 (HOX), or the combination of the two. There was a significant decrease in the receptor mRNA levels in cells transfected with HOXA10 siRNA (siRNA). Simultaneous treatment with P and HOXA10 siRNA (siRNA + Prog) blocked the P effect on subunit expression. *P < 0.05 vs. control (CTL); **P < 0.01 vs. P.

Fig. 3.

Immunocytochemical visualization of GABA_A π receptor localization on confocal laser microscopy and regulation by progesterone. Cell periphery was stained with Texas red against cytokeratin 5/6/18. GABA_A π receptors were stained with fluorescein. A: vehicle control-treated cells with nonlocalized receptors. B: treatment with progesterone leads to an increase in localization of the receptor to the cell membrane. C: treatment of the cells with progesterone and HOXA10 siRNA prevents peripheral translocation of the receptors.

Fig. 4.

Immunocytochemical visualization of GABA_A π receptor localization on confocal laser microscopy. Cell periphery was stained with Texas red, using an antibody against cytokeratin 5/6/18. GABA_A π receptors were stained with fluorescein. The nucleus was stained with Topro-3. Cells were transfected with pcDNA-HOXA10, leading to greatly enhanced peripheral localization of the receptor.

Fig. 5.

Quantification of GABA_A π receptor peripheral localization. Displayed are the mean numbers of cells with peripheralization of the receptor per field. Treatment with P (P = 0.002) or transfection with HOX (P = 0.001) each led to significantly increased peripheralization. The P-induced peripheralization was blocked with HOXA10 siRNA (siRNA), indicating a role for HOXA10 in mediating the effect of P. *P < 0.01 vs. CTL; **P < 0.01 vs. P treatment. HPF, high-power field.